The broad objectives of this proposal are to determine how entry and exit from mitosis is controlled, how centrosomes and kinetochores function and interact to form the spindle, and how the forces for chromosome motion & positioning are generated & regulated. In Aim #1 time-lapse video lightmicroscopy (VLM), GFP-imaging and 3-D electron microscopy (3D-EM) will be used to: a) test the hypothesis that microtubulesare involved in the checkpoint regulating progression through prophase in vertebrates; & to determine b) if prophase cells, induced by hypothermia to revert to 62, fail to re-enter mitosis after 24 hrs because they lack cyclin A or Cdc25; & c) the etiology of chromosome segregation abnormalities in such revertants when induced to re-enter mitosis after 16 hrs in G2. In Aim #2 we will use cell fusion, GFP-imaging, 3D EM and antibody injection to evaluate the hypotheses that in vertebrate cells: a) the numbers & length of astral microtubules, formed by centrosomes not associated with chromosomes, changes during prometaphase; b) that separating asters interact during prometaphase in the absence of associated chromosomes; c) that the acentrosomal pathway for spindleassembly is a constitutive component of mitosis; & d) that centrosomes &/or centrioles are required for the completion of cytokinesis. In Aim #3 we will use antibody injection, VLM and 3D-EM to test the hypotheses that: a) the kinetochore associated motor responsiblefor fast poleward motion during kinetochore attachment is no longer functional during anaphase, and b) that ZW-10 is involved in kinetochore re-orientation during mitosis & meiosis in Drosophila. In Aim #4 we will use laser microsurgery to determine: a) if "polar winds" are present during meiosis & mitosis in Drosophilaspermatocytes & neuroblasts& if so whether they are present in neuroblasts lacking functional Klp38b &/orNod proteins. We will also test the hypotheses that: b) destroying part of one kinetochore does not inhibit chromosome congression in vertebrates, & c) that a chromosome, bioriented on a spindle formed from two poles that nucleate vastly different numbers of microtubules, congresses closer to the weak spindle pole. Finally, in Aim #5 we will use laser microsurgery and GFP-imagingto test the hypotheses: a) that the mitotic arrest induced in vertebrate cells by nocodazole or Taxol is due to kinetochores; b) that C-anaphase in these cells requires a functional centrosome; c) that C-anaphase is correlated temporally with the destruction of cyclin B; & d) that Drosophila spermatocytes lack a spindle assembly checkpoint. The knowledgeobtained from these studies is required to better understand the etiology of various birth defect syndromes and cancers, to design new therapeutic strategies for the control of cell proliferation, and for the treatment of other disease states involvingmicrotubules including arthritis, metastasis and Alzheimer's.